Solid-state Light Emitting Devices (LEDs), such as light emitting diodes or solid state lasers, are now commercially available with increasingly higher power density in the visible and near ultraviolet (UV) spectrum. Some of these LEDs, and particularly high brightness UV and visible light emitting diodes, now have demonstrated capability for supplanting high power lamps (e.g. mercury lamps) and conventional laser-based systems, as light sources for fluorescence microscopy and other spectroscopic applications. LED light sources are relatively compact, are electronically controllable, can be modulated to provide pulsed operation and potentially have advantages with respect to cost and relatively long lifetime.
In a typical fluorescence microscopy application, for example, for biomedical diagnostics, a biological sample comprising a fluorophore is illuminated with light of a known wavelength, and the resulting fluorescence is observed using a CCD camera. While it used to be sufficient to illuminate and capture fluorescence in a qualitative manner, an increasing drive towards quantitative analysis has necessitated more accurate control over experimental conditions and parameters. In some systems, the illumination system may comprise several LED light sources, each producing light of a different wavelength or colour, coupled to the microscope system for pulsed excitation of fluorescence from a biological sample. Existing LED illumination systems provide only limited flexibility to control light pulses, or pulse trains from multiple LEDs and/or to synchronize with external devices, such as radiometers, detectors, cameras or other microscope peripherals.
For example, in some applications, during an experiment it may be desirable to expose cells to a light pulse of controlled duration and intensity to minimize photo-bleaching of cells. To that end, specific and precise control of the light source pulses is required together with triggering of external devices, in advance of, or delayed, relative to turning on or off of the light source. Typically, tight and flexible control of the LED light source in a time range from a few microseconds to many hours may be required, with adjustability in the microsecond, millisecond or second ranges.
Thus, a typical fluorescence microscopy experiment may require the following generic procedure to be followed:                Activate LED(s) of a selected wavelength(s) to predetermined intensity level(s),        Acquire an image frame from a digital camera,        Deactivate LED(s),        Repeat sequence if necessary.        
The complexity of the experiment may vary, for example, depending on the number of LEDs being turned on/off, the acquisition time of a peripheral device, such as a CCD camera, the sequencing of the various LEDs, the equipment synchronization, the repetition rate, as well other parameters and controls.
The requirement for triggering stems from the need to align events in time between multiple physical elements in a system. That is, a detector or CCD camera, for example, may need to be triggered to start acquiring data at a specific time relative to when the experiment was started.
Many known LED sources allow for external pulse generating equipment to be connected to the LED light source for automation of LED activation/deactivation during a fluorescence experiment. However, such equipment typically requires BNC cable connectivity for each LED and a minimum of 1 external pulse generator on the bench. A separate signal must be sent to trigger the camera exposure. This may either be derived from the pulse itself or from the signal generator trigger output. In situations where there are different pulse lengths and repetition rates on each LED, there would be a requirement for individual pulse generators and associated cabling for each unique pulse signature being used.
The ability to generate pulses internally would remove the need for external pulse generators, external trigger derivation and associated cabling complexity. However, existing LED light sources do not provide individual pulse control for multiple LEDs. One commercially available system operates in either internal pulse generation mode or external pulse generation mode. Such a limitation means that the system is either pulsed externally or relies on the internal generator. Although individual LEDs can be turned on or off, the consequence of this is that the pulses are phase aligned, copied across all the LEDs. Significant additional hardware, including an additional external pulse generator, is required for fluorescence experiments where there is a requirement for the LEDs to be activated and deactivated in sequences which are markedly out of phase and/or with different pulse characteristics.
For example, referring to FIG. 1 (Prior Art), which illustrates light pulse sequences when a single internal or external pulse generator is used to control multiple LEDs, e.g. LED1 and LED2, all LEDs in the system will be on or off at the same time and with the same duty cycle. There is only one trigger indicating when all LEDs should turn on and off. There is no independent channel adjustment. Referring to FIG. 2 (Prior Art) illustrating light pulse sequences obtained by using multiple pulse generators to provide independent control of LEDs, each LED can have a unique on/off time. However, the delay or phase shift between the first and second pulse trains is difficult to control.
The lack of integrated pulse control functionality in known system results in significant additional equipment cost for external pulse generators and control equipment, and resultant associated bench space and cabling requirements. Even for skilled users, such an arrangement also results in significant experimental set-up time and complexity for experiments requiring control of light pulse characteristics and relative phase for multiple LEDs. A lower cost system and simpler set-up and operation would be desirable.
Thus, there is a need for improved systems, methods and devices for control of pulse generation and synchronization of LED light sources, for applications such as fluorescence microscopy.